SUMMARY Nave lymphocytes, upon receiving appropriate co-stimulatory signals, become activated and go through a rapid proliferative burst before assuming effector functions directed at killing the target ?invading? organism or cancer cell. This rapid proliferative burst requires increased uptake and utilization of glucose in anabolic pathways that produce building blocks for biomass. To support proliferation activated T cells switch to glutamine as the carbon source for oxidative phosphorylation (OXPHOS). The shift to aerobic glycolysis and enhanced dependence on glutamine in activated T cells is well documented, but the signaling pathways that facilitate this shift are poorly understood. Recent studies from our group point to a key role for human Bcl-2 family BH3-only protein, Noxa, in controlling this activation-induced metabolic switch in T cells. BH3-only proteins are by definition pro-apoptotic, but Noxa exhibits a pro-growth and survival function as well, in leukemia cells. This non-canonical function of Noxa is also evident in primary human T lymphocytes. Noxa is significantly upregulated in T cells following TCR engagement, and remains high as the activated cells proliferate. Additionally, it requires constant availability of glutamine to remain highly expressed. Early studies suggest that Noxa plays an essential role in T cell activation and the metabolic reprogramming associated with activation. We will test a central hypothesis that states - Noxa is required for the metabolic switch to glutaminolysis in activated human T cells and for their differentiation to the effector phenotype. The first aim will investigate the role of Noxa in T cell activation and glutamine metabolism. We will determine whether glutamine mediates the induction of Noxa expression in T cells through regulation of microRNAs. This aim will also test the hypothesis that Noxa is required for glutaminolysis and for proliferation of activated T cells using novel CRISPR-generated Noxa KO primary T cells, biochemical assays, metabolic phenotyping, and metabolomics. Although loss of Noxa impairs glutaminolysis, it does not affect mitochondrial fitness suggesting stimulated T cells shift to an alternative fuel source for oxidative phosphorylation. In Aim 2 we will use multi- parameter flow cytometry, and metabolic and functional assays to test the hypothesis that Noxa enables T effector cell generation by promoting glutaminolysis, and prevents differentiation of T cells to a regulatory (Treg) or memory (TM) phenotype by suppressing fatty acid oxidation. An understanding of the metabolic needs of T cells as they undergo expansion will help to develop therapeutic strategies targeted at metabolism. This research could reveal avenues to exploit the crosstalk between Noxa and glutamine metabolism to better understand cellular events that drive the metabolic switch in T cells. The proposed work also will offer insights into Noxa?s role in driving differentiation of T cells into effector, regulatory or memory subtypes and help evaluate the protein?s potential as a therapeutic tool.